The innate immune regulator MyD88 dampens fibrosis during zebrafish heart regeneration

The innate immune response is triggered rapidly after injury and its spatiotemporal dynamics are critical for regeneration; however, many questions remain about its exact role. Here we show that MyD88, a key component of the innate immune response, controls not only the inflammatory but also the fibrotic response during zebrafish cardiac regeneration. We find in cryoinjured myd88−/− ventricles a significant reduction in neutrophil and macrophage numbers and the expansion of a collagen-rich endocardial population. Further analyses reveal compromised PI3K/AKT pathway activation in the myd88−/− endocardium and increased myofibroblasts and scarring. Notably, endothelial-specific overexpression of myd88 reverses these neutrophil, fibrotic and scarring phenotypes. Mechanistically, we identify the endocardial-derived chemokine gene cxcl18b as a target of the MyD88 signaling pathway, and using loss-of-function and gain-of-function tools, we show that it controls neutrophil recruitment. Altogether, these findings shed light on the pivotal role of MyD88 in modulating inflammation and fibrosis during tissue regeneration.

The innate immune response is triggered rapidly after injury and its spatiotemporal dynamics are critical for regeneration; however, many questions remain about its exact role.Here we show that MyD88, a key component of the innate immune response, controls not only the inflammatory but also the fibrotic response during zebrafish cardiac regeneration.We find in cryoinjured myd88 −/− ventricles a significant reduction in neutrophil and macrophage numbers and the expansion of a collagen-rich endocardial population.Further analyses reveal compromised PI3K/AKT pathway activation in the myd88 −/− endocardium and increased myofibroblasts and scarring.Notably, endothelial-specific overexpression of myd88 reverses these neutrophil, fibrotic and scarring phenotypes.Mechanistically, we identify the endocardial-derived chemokine gene cxcl18b as a target of the MyD88 signaling pathway, and using loss-of-function and gain-of-function tools, we show that it controls neutrophil recruitment.Altogether, these findings shed light on the pivotal role of MyD88 in modulating inflammation and fibrosis during tissue regeneration.
Myocardial infarction is the most common cause of cardiac injury in humans, often leading to an irreversible loss of heart muscle tissue 1,2 .Unlike the adult mammalian heart, the zebrafish heart can regenerate lost tissue upon different types of injuries, making it a powerful animal model to study heart regeneration [3][4][5][6] .
Immediately after cardiac injury, tissue damage triggers the activation of a sterile inflammatory response, with immune cells infiltrating the injured area, clearing dead cells and debris, modifying the extracellular matrix (ECM) and initiating signaling cascades 7,8 .As shown previously, this response is programmed to be resolved timely and is essential for triggering the regenerative response in both zebrafish [9][10][11] and neonatal mice 12 .On the other hand, in the non-regenerative adult mammalian heart, inflammation persists longer and has been linked with tissue damage after myocardial infarction 13,14 .Thus, it is essential that the inflammatory response is tightly controlled to allow for successful regeneration 14 .
Cardiac inflammation after injury involves the release of damageassociated molecular pattern (DAMP) molecules from dying and stressed cells, which bind to Toll-like receptors (TLRs) [15][16][17] .Importantly, most TLRs, as well as interleukin-1 receptors (IL-1Rs), signal through the same adaptor molecule, myeloid differentiation factor 88 (MyD88) 18,19 .While the MyD88 signaling pathway is involved in the activation of the Article https://doi.org/10.1038/s44161-024-00538-5during cardiac regeneration in zebrafish, we dissociated cryoinjured ventricles from myd88 LOF mutants (myd88 −/− ) 32 and wild-type (WT) (myd88 +/+ ) siblings, sorted live cells and performed single-cell RNA sequencing (scRNA-seq) analysis (Fig. 1a and Supplementary Fig. 1a).We focused our analysis at the early time point of 24 hours post cryoinjury (hpci), when myd88 expression peaks in the cryoinjured zebrafish heart (Supplementary Fig. 1b) 10 .In addition, as the receptors of the MyD88 signaling pathway can rapidly recognize cardiac tissue damage through binding their ligands, 24 hpci is a good time point because it is within the early acute inflammatory phase 33 .We performed quality control analysis, applied filtering cutoffs and obtained a dataset consisting of 11,735 cells (8,181 and 3,554 myd88 +/+ and myd88 −/− cells, respectively).To assess cell type diversity, we took into consideration known marker genes and identified five major cell cluster populations, including endocardial cells, coronary endothelial cells (cECs), myeloid cells, epicardial and epicardium-derived cells (EPDCs) and mesenchymal cells (Fig. 1b,c) [34][35][36] .We did not detect a CM population, presumably because their large size was incompatible with our sample preparation pipeline as reported previously 36 .As myeloid cells activate the MyD88 signaling pathway 10,22,37,38 , we decided to perform unbiased subclustering of the myeloid population to investigate their diversity (Fig. 1d).This analysis revealed five distinct myeloid clusters, of which four expressed macrophage marker genes (for example, mpeg1.1, c1qa) at higher levels and one expressed neutrophil marker genes (for example, mpx, lyz) at higher levels (Fig. 1d and Extended Data Fig. 1a,b).We identified additional genes highly expressed in this presumed neutrophil cluster and indeed observed their enrichment in neutrophils in published datasets (Extended Data Fig. 1c) [34][35][36] .Comparison of the myeloid populations between myd88 +/+ and myd88 −/− ventricles revealed pronounced differences, such as a strong reduction of the neutrophil and the marco macrophage clusters in myd88 −/− ventricles (Fig. 1d).We also observed that the expression of inflammatory genes was increased in these two clusters, suggesting that they contain pro-inflammatory cells (Fig. 1e).myd88 −/− ventricles also exhibited expansion of a macrophage subpopulation that is transcriptionally unique (enriched genes: gpx1a, mrc1b, timp4.2,lxn, lta4h, csf3r, cfh) and is not present in myd88 +/+ ventricles (Extended Data Fig. 1d,e).
Neutrophils are known pro-inflammatory mediators and immediate responders to tissue damage [39][40][41] .They can shape the regenerative outcome because they influence important regeneration events, including clearance of dead cells and debris from the injured area, macrophage polarization, effective revascularization, epicardial activation and myocardial regeneration [42][43][44][45][46][47] .The MyD88 signaling axis, once activated, facilitates neutrophil recruitment 22,23,37,48 .To further investigate the neutrophil phenotype observed in cryoinjured myd88 −/− ventricles (Fig. 1d), we used the TgBAC(mpx:GFP) neutrophil reporter line to determine neutrophil numbers after injury.While at 6 hpci we did not observe obvious differences between myd88 −/− and myd88 +/+ sibling ventricles (Extended Data Fig. 1f,g), at 24 hpci neutrophil numbers were significantly reduced in myd88 −/− ventricles and their numbers immune response, a comprehensive understanding of its role in cardiac repair and regeneration remains elusive, with studies reporting conflicting results 14,20,21 .As excessive and persistent inflammation has been linked with adverse regeneration outcomes, studies have often focused on blocking MyD88 or TLR function to restrict inflammation.Limiting inflammation in MyD88 or TLR loss-of-function (LOF) mouse models improved their regenerative potential [22][23][24] .Conversely, TLR4-MyD88 activation in mesenchymal stem cells conferred cardioprotective effects in cardiac ischemia-reperfusion injury models in mouse 25 , TLR4-MyD88 activation in cardiomyocytes (CMs) reduced their apoptosis in vitro 26 , and MyD88 cardioprotective effects were highlighted in a rat aortic banding model 27 .The critical role of MyD88 signaling in preserving cardiac function and limiting progression to heart failure was also shown in a dominant negative MyD88 transgenic mouse model 28 .Depletion of MyD88 in T cells results in increased inflammation and fibrosis after transverse aortic constriction in mouse 29 .While most of these studies were carried out in non-regenerative mammalian models, understanding the role of MyD88 in a regenerative system should provide insights into the pathogenesis of cardiovascular diseases and the development of new treatment approaches.In addition, the precise roles of the MyD88 signaling axis beyond cardiovascular inflammation and its contribution to other responses necessary for regeneration have so far been largely overlooked.Therefore, we directed our study toward unraveling the cell-specific functions of MyD88 signaling during cardiac regeneration in zebrafish.
In this study, we use newly generated genetic tools and transcriptomic profiling to investigate the role of MyD88 signaling during zebrafish cardiac regeneration at several stages and across different cell types; we find that MyD88 not only has a pivotal role in the immune response, but also a previously unidentified function in limiting endothelial-mediated fibrosis.Specifically, we show that in addition to its role in neutrophil and macrophage recruitment, MyD88 is important to limit a fibrotic response by regulating the transcriptome of endocardial and mesenchymal cells and by limiting myofibroblast numbers, fibrin levels and scar sizes.Furthermore, we identify a critical role for MyD88 signaling in regulating the phosphoinositide 3 kinase (PI3K)/AKT pathway in the injured endocardium.Notably, using an endothelial-specific mutant rescue strategy, we reveal the essential role of MyD88 in endothelial cells to promote neutrophil recruitment and limit the fibrotic response.Mechanistically, we identify the endocardial chemokine gene cxcl18b as a transcriptional target of the MyD88 signaling pathway and showed that it controls neutrophil recruitment.

Reduced inflammatory cell numbers in injured myd88 −/− ventricles
Genes encoding MyD88 and MyD88 pathway-related signaling components are expressed in a wide range of tissues and cell populations even in the absence of tissue damage and infection 15,30,31 .To begin to understand how MyD88 signaling affects different cell populations  remained reduced at least until 96 hpci (Fig. 1f,g).We also counted the number of neutrophils at 6 days post sham (dps) surgery and 7 days post cryoinjury (dpci), but did not observe any significant differences at these time points (Extended Data Fig. 1f,g).Together these data indicate that neutrophil numbers were reduced at 24 and 96 hpci in myd88 −/− ventricles.
As with neutrophils, the MyD88 signaling axis contributes to macrophage recruitment 10,37,38,49 .Thus, we used the Tg(mpeg1:EGFP)      macrophage reporter line to determine whether macrophage numbers were also affected in cryoinjured myd88 −/− ventricles.Both scRNA-seq analysis and immunostaining for mpeg1:EGFP + cells revealed that total macrophage numbers were not affected in myd88 −/− ventricles at 24 hpci (Fig. 1h,i and Extended Data Fig. 1h).While we did not observe a significant reduction in total macrophage numbers at 24 hpci, our transcriptomic data showed that some macrophage populations might be affected, with the marco macrophage population being severely reduced and the hp-1 macrophage population clearly increased in myd88 −/− ventricles (Fig. 1d and Extended Data Fig. 1d,e).As the macrophage response initiates and develops later than the neutrophil response after injury, we set out to analyze cryoinjured ventricles at a later time point, when a macrophage phenotype might be more obvious.To this end, we selected 96 hpci, a time point when macrophage numbers increases significantly in WT zebrafish 10,34,50,51 , and indeed found a reduction in mpeg1:EGFP + cells in myd88 −/− ventricles compared with myd88 +/+ siblings (Fig. 1h,i).
Taken together, these transcriptomic analyses and immunostaining data reveal that during zebrafish cardiac regeneration, MyD88 is important for neutrophil and macrophage recruitment and for enhancing the inflammatory state of immune cells.

MyD88 signaling attenuates fibrosis in injured ventricles
Besides analyzing myeloid cells, the most extensively studied activators of the MyD88 signaling pathway 10,22,37,38 , we wanted to determine which other cell types were important for MyD88 function during cardiac regeneration.As reported previously, endothelial cells express myd88 and components of the MyD88 signaling axis, such as the tlr genes 52,53 .To investigate the state and role of endocardial cells in the absence of MyD88 function during regeneration, we performed unbiased subclustering analysis of the endocardial population in our scRNA-seq dataset.This analysis revealed the presence of four distinct endocardial clusters, represented in different proportions in cryoinjured myd88 −/− and myd88 +/+ ventricles (Fig. 2a).Specifically, the most abundant endocardial cell cluster found in myd88 +/+ ventricles (irx5a endocardial cells) was significantly reduced in myd88 −/− ventricles.Interestingly, the most abundant endocardial cluster in myd88 −/− ventricles displayed high expression levels of genes encoding collagens (col1a2, col1a1a, col5a1, col1a1b, col5a2a, col6a2, col6a1) and genes associated with fibrosis (postna, gstm.3,sparc, acta2) 34,54 .Hence, we annotated this cluster as 'collagen-rich endocardial cells' (Fig. 2a,b).Pseudotime trajectory analysis indicated that cells transition from the wound endocardial cluster serpine1 (ref.55) to the other endocardial clusters (Extended Data Fig. 2a).To investigate the relationship between endocardial clusters, we also performed velocity analysis.The resulting data indicate that endocardial cells in myd88 −/− ventricles tend to progress toward the collagen-rich endocardial state (Extended Data Fig. 2b).As shown previously in mouse [56][57][58] and zebrafish 35,59 , the endocardium contributes to the activated fibroblast cell population and to α-smooth muscle actin (αSMA) + myofibroblasts in injured hearts; thus, increased expression of collagen and fibrotic genes in the cryoinjured myd88 −/− endocardium (Fig. 2a,b) could reflect an increase in these endocardium-derived cells.αSMA + myofibroblasts are the key contributors to ECM fibrotic remodeling and scar formation in the injured heart 56,58,60 .Moreover, in the context of MyD88 signaling, MyD88 depletion in T cells results in increased cardiac fibroblast to myofibroblast transformation in vitro, as indicated by elevated αSMA and collagen type 1 expression in fibroblasts 29 .To investigate the role of MyD88 in myofibroblast differentiation during zebrafish cardiac regeneration, we immunostained for αSMA expression on sections of cryoinjured ventricles carrying the ET(krt4:EGFP) endocardial enhancer trap line (Fig. 2c).At 96 hpci, we found an increased abundance of αSMA + cells in myd88 −/− compared with myd88 +/+ siblings (Fig. 2c,d), which remained elevated until 7 dpci (Extended Data Fig. 2c,d).To differentiate between αSMA + cells of different origins, we quantified intraventricularly localized αSMA + cells, presumably of endocardial and fibroblast origin, and excluded superficially localized αSMA + cells, presumably of epicardial, fibroblast and perivascular origin 35,61 .In line with the fibrotic transcriptomic profile of the myd88 −/− endocardium (Fig. 2a,b), at 96 hpci we found an increased number of intraventricular αSMA + cells in myd88 −/− zebrafish compared with myd88 +/+ siblings (Fig. 2d).Superficially localized αSMA + cells were also elevated in cryoinjured myd88 −/− ventricles (Extended Data Fig. 2e), but not to the same extent as the intraventricularly localized αSMA + cells.Altogether, these data indicate that MyD88 suppresses myofibroblast differentiation during zebrafish cardiac regeneration.
Activation of MyD88 signaling in mesenchymal stem cells reportedly conferred cardioprotective effects via Stat3 in a mouse model of cardiac ischemia-reperfusion injury 25 .As myofibroblasts, a mesenchymal cell type, are affected in myd88 −/− ventricles, we decided to take a closer look at the mesenchymal population in our scRNA-seq dataset.Unbiased subclustering analysis indicated the presence of three mesenchymal subclusters.Strikingly, strong differences between the myd88 −/− and myd88 +/+ samples resulted in one cluster (hapln1a mesenchymal cells) being of almost completely myd88 −/− and another (cst14a.1 mesenchymal cells) being of almost completely myd88 +/+ identity (Extended Data Fig. 2f).Specifically, the myd88 −/− hapln1a mesenchymal cell cluster exhibited increased expression of endothelial-to-mesenchymal transition and fibrotic-related genes, such as sox9a and acta2 (Extended Data Fig. 2g,h) 59 .
As our data pointed to an endocardial-related fibrotic phenotype in cryoinjured myd88 −/− ventricles, we next analyzed additional endocardial processes that could be affected in the absence of myd88 function.Endocardial hyperinvasion of the injured area has been linked with increased endothelial-to-mesenchymal transition and fibrotic remodeling 59 .However, we did not observe any obvious differences between myd88 −/− and myd88 +/+ siblings with respect to the krt4:EGFP + and Cdh5 + endocardial cell area covering the injury at 96 hpci (Extended Data Fig. 3a,b) or 7 dpci (Extended Data Fig. 3c,d), respectively.Additionally, the Aldh1a2 activation pattern was similar in myd88 −/− and myd88 +/+ sibling ventricles at 96 hpci (Extended Data Fig. 3a) 55 , indicating that only endothelial-to-mesenchymal transition processes were affected in cryoinjured myd88 −/− ventricles.
Blocking MyD88 signaling in uninjured murine hearts leads to increased fibrosis 28 .To investigate the regenerative potential and scar resolution abilities of cryoinjured myd88 −/− ventricles, we performed acid fuchsin orange G (AFOG) staining.As reported previously, scar tissue in 14 dpci WT hearts consists of an extensive collagen network and a ring-like peripheral fibrin structure 5,33,62 .In contrast, the scar tissue in myd88 −/− ventricles at 14 dpci displayed thicker fibrin-rich areas in comparison with their myd88 +/+ siblings (Fig. 2e), and these increased fibrin levels persisted until at least until 30 dpci (Fig. 2e-g).However, the collagen proportions within the scars remained similar between myd88 −/− and myd88 +/+ siblings (Extended Data Fig. 4a).Additionally, at 30 dpci, there were more myd88 −/− ventricles with bigger scar areas compared with their myd88 +/+ siblings (Extended Data Fig. 4b).We also examined scars at 90 dpci to analyze fibrotic tissue persistence, but did not observe significant differences at this time point (Extended Data Fig. 4c,d).
In summary, the fibrotic-like transcriptomic profile (enrichment in collagen and fibrotic genes) observed in both endocardial and mesenchymal cells, along with the increased number of total and intraventricular αSMA + cells, increased fibrin levels and larger scars in cryoinjured myd88 −/− hearts, collectively indicate that MyD88 signaling has a role in limiting fibrosis during zebrafish cardiac regeneration.

Article
https://doi.org/10.1038/s44161-024-00538-5line at 96 hpci and conducted bulk RNA-seq analysis (Fig. 3a and Supplementary Fig. 2).In line with the previously observed fibrotic phenotype (Fig. 2), gene set enrichment analysis (GSEA) revealed an increase in the regulation of the cellular response to transforming growth factor-β stimulus in the cryoinjured myd88 −/− endocardium (Extended Data Fig. 5a).Additionally, GSEA (Extended Data Fig. 5a) and closer examination of the most differentially expressed genes (Fig. 3b) revealed that immune-associated processes, such as TLR, nucleotide-binding oligomerization domain (NOD)-like receptor, C-type lectin receptor and cytosolic DNA-sensing signaling pathways, as well as the mitogen-activated protein kinase (MAPK) signaling pathway, were impaired in the myd88 −/− endocardium.As the MAPK signaling pathway has been linked with the activated endocardium in both the cardiac resection 63,64 and cryoinjury 51 models, we immunostained for phosphoERK (pERK), an indicator of activated MAPK signaling, in ET(krt4:EGFP) ventricles and verified its presence in the endocardium at 96 hpci.However, we did not observe statistically significant differences in the pERK + cell area covering the injury between myd88 −/− and myd88 +/+ siblings (Extended Data Fig. 6a,b), indicating that defective MAPK signaling was not responsible for the endocardial phenotype observed in myd88 −/− ventricles.This observation is in line with a previous study where lipopolysaccharide treated Myd88 −/− mice displayed activation of the MAPK signaling pathway 65 .GSEA and examination of the most differentially expressed genes (DEGs) (Fig. 3b, Supplementary Table 1 and Extended Data Fig. 5a,b) further indicated that activation of the PI3K/AKT pathway is impaired in the myd88 −/− endocardium.Additionally, GSEA terms related to upstream (ErBB, IGF, VEGF, FGF and NGF pathways) and downstream components (mTOR pathway) of PI3K/AKT signaling were substantially downregulated in the cryoinjured myd88 −/− endocardium (Extended Data Fig. 5a,b) 64,[66][67][68][69] .Interestingly, MyD88 signaling can also regulate PI3K/AKT pathway activation 28,70,71 .Hence, we costained for phospho-Akt (pAkt), which indicates activation of PI3K/AKT signaling when present in the nucleus, together with the nuclear endothelial cell marker Fli1 in sections of ET(krt4:EGFP) ventricles and found a tendency for reduced activation of the PI3K/AKT pathway in myd88 −/− endocardial cells at 96 hpci (Fig. 3c,d).Given that PI3K/AKT activation affects cell proliferation 68,72 , we then assessed endocardial proliferation at 96 hpci, when it reaches high levels in WT 55,68 .Notably, we found significantly reduced proliferation in myd88 −/− endocardial cells at 96 hpci (Fig. 3e,f).
In summary, these findings underscore the critical role of MyD88 in regulating signaling cascades in the injured endocardium.Particularly, we identified impairments in the immune response, in the activation of the PI3K/AKT pathway and in endocardial cell proliferation in cryoinjured myd88 −/− ventricles.

MyD88 signaling promotes revascularization and CM repopulation
Revascularization is one of the earliest processes observed in the injured area and it is vital for effective cardiac tissue regeneration 73,74 .Following cryoinjury of the zebrafish ventricle, cECs undergo proliferation and initiate the sprouting of new vessels, both superficially and intraventricularly.While intraventricular revascularization is orchestrated by endocardial vascular endothelial growth factor A signaling, superficial revascularization is partially regulated by Apelin signaling 74 .We found that both endocardial VEGF and Apelin signaling pathways were impaired in cryoinjured myd88 −/− ventricles (Extended Data Fig. 5a).Additionally, neutrophils, which were significantly reduced in myd88 −/− ventricles (Fig. 1d,f,g), express VEGF and promote revascularization 42,44,46 .Hence, we used the Tg(-0.8flt1:RFP)reporter line, which labels cECs and assessed their proliferation at 96 hpci, when cEC proliferation peaks in cryoinjured WT ventricles 74 .We found that cEC proliferation was significantly reduced at 96 hpci in myd88 −/− hearts when compared with myd88 +/+ siblings (Extended Data Fig. 7a,b).While cEC proliferation recovered by 7 dpci (Extended Data Fig. 7c,d), coronary vessel coverage in the injured area remained impaired at 7 dpci (Extended Data Fig. 7e,f) in myd88 −/− .Together, these data reveal that revascularization of the injured area was affected in cryoinjured myd88 −/− ventricles.
During cardiac regeneration in zebrafish, CMs undergo dedifferentiation and proliferation to repopulate the injured tissue [75][76][77] and this process is heavily influenced by the microenvironment surrounding the CMs 78 .Given the altered inflammatory and fibrotic environment in cryoinjured myd88 −/− ventricles, along with the impaired numbers and functions of various cell types that precede CM appearance in the injured area, we decided to assess CM behavior.We observed a significant reduction in CM proliferation in the injury border zone at 96 hpci and 7 dpci in myd88 −/− cryoinjured ventricles (Fig. 4a-d).We also examined CM proliferation at 14 dpci and dedifferentiation at 96 hpci 79 but did not find any significant differences (Extended Data Fig. 8a-d).Furthermore, we quantified CM protrusive activity toward the injury at 72 hpci and 7 dpci and found that while the number of protrusions was the same between myd88 −/− and myd88 +/+ siblings, CM protrusions were shorter in myd88 −/− ventricles at 7 dpci (Fig. 4e-g).These results highlight the essential role of MyD88 in facilitating efficient CM proliferation and protrusion toward the injured tissue during cardiac regeneration.

Endothelial myd88 overexpression reverses myd88 −/− phenotypes
To investigate whether the loss of MyD88 in endothelial cells was the contributing factor for the fibrotic phenotype observed in cryoinjured myd88 −/− ventricles, we specifically overexpressed (OE) myd88 in endothelial cells.For this purpose, we generated a Tg( fli1a:myd88,EGFP) line that expresses myd88 and EGFP under the control of a bidirectional fli1a promoter.As expected, myd88 expression was increased in transgenic zebrafish when compared with non-transgenic sibling larvae and adult cryoinjured ventricles (Extended Data Fig. 9a), thereby validating the overexpression ability of the tool.In addition, the transgenic EGFP signal colocalized with the endocardial Aldh1a2 signal in cryoinjured ventricles, validating the endothelial-specific expression profile of the Tg( fli1a:myd88,EGFP) line (Extended Data Fig. 9b).We analyzed myd88 −/− and myd88 +/+ siblings carrying the overexpression transgene and found that the previously elevated αSMA + cells (total and intraventricular) (Fig. 2c,d) not only reverted to control levels, but also significantly decreased in cryoinjured myd88 −/− ventricles at 96 hpci (Fig. 5a,b).To assess whether endothelial-specific myd88 overexpression could improve the regeneration potential of cryoinjured myd88 −/− ventricles, we analyzed scars at 30 dpci.Remarkably, Tg( fli1a:myd88,EGFP); myd88 −/− and myd88 +/+ sibling ventricles were indistinguishable in terms of fibrin abundance and scar area size (Fig. 5c-f).Altogether, these data further indicate that endothelial MyD88 function is critical to limit fibrosis in the regenerating zebrafish heart.
Endothelial cells activate immune-regulatory programs, secrete cytokines and chemokines, and facilitate early immune cell recruitment in response to cardiac injury 14,52,55,80 .They also express MyD88 and components of the MyD88 signaling axis, including TLRs 52,53 .To determine whether myd88 overexpression in endothelial cells could also restore the low neutrophil levels observed in cryoinjured myd88 −/− ventricles (Fig. 1d,f,g), we quantified the number of Mpx + cells and found that neutrophil levels were indeed restored at 96 hpci (Fig. 5g,h), indicating that MyD88 function in endothelial cells is sufficient to rescue the neutrophil phenotype.Overall, these endothelial-specific myd88 overexpression experiments support the model that endothelial-specific MyD88 activation promotes neutrophil recruitment in the cryoinjured heart.

MyD88-Cxcl18b signaling promotes neutrophil recruitment after cardiac injury
To gain a deeper understanding of the targets of the MyD88 signaling pathway during zebrafish cardiac regeneration, we conducted
Importantly, a previous study reported that cxcl18b upregulation is downstream of MyD88 signaling in infection models 32 ; however, the specific role of Cxcl18b during cardiac regeneration remains to be elucidated.Regarding its expression pattern, cxcl18b is expressed in caudal hematopoietic and endothelial cells in infected zebrafish larvae 83 and in endocardial cells in regenerating zebrafish hearts 34 .We used a Tg(cxcl18b:EGFP) reporter line 83 to look more closely at cxcl18b expression and observed clear EGFP expression in endocardial and epicardial cells at 24 hpci (Extended Data Fig. 10a).
To understand how loss of Cxcl18b might affect neutrophil recruitment during cardiac regeneration, we generated a cxcl18b mutant allele using the CRISPR-Cas9 technology.As many chemokines have high sequence homology and to avoid transcriptional adaptation events 87,88 , we specifically generated a full locus deletion allele (Extended Data Fig. 10e).To assess cxcl18b expression levels, we performed larval fin fold amputations, which trigger cxcl18b upregulation in WT cells 84,85 , and collected larvae 6 hours post amputation (hpa) for RT-qPCR analysis.As anticipated, cxcl18b full locus deletion mutants completely lacked cxcl18b expression (Extended Data Fig. 10f).To examine whether lack of Cxcl18b could affect neutrophil recruitment in cryoinjured ventricles, we quantified neutrophil abundance at 24 and 96 hpci.Notably, while the Mpx + cell count was unaffected at 24 hpci, there was a significant difference between cxcl18b −/− and cxcl18b +/+ siblings at 96 hpci (Fig. 6g,h).
In summary, these findings together indicate that Cxcl18b is a chemokine whose expression is upregulated upon cardiac cryoinjury by MyD88 and is at least partly responsible for neutrophil recruitment to the injured area.

Discussion
After cardiac injury, the zebrafish heart mounts a robust innate immune response, which is crucial for the regeneration process [9][10][11] .The precise regulation of this early immune response is essential, underscoring the need for a detailed understanding of its regulators and underlying mechanisms 14 .Key activators of this response are the TLR and IL-1R signaling pathways, which signal predominantly through the adapter protein MyD88 (refs.16-19).While previous studies mostly focused on immune cells to investigate MyD88 function, in this study we examined other cell types of the heart and identified an unexplored function for MyD88: not only does it regulate the inflammatory response, but it also limits the endocardial fibrotic response to injury.Specifically, we observed a significant reduction in pro-inflammatory neutrophil and macrophage populations in cryoinjured myd88 −/− ventricles.More surprisingly, we also observed in myd88 −/− ventricles (1) the expansion of a collagen-rich endocardial population, (2) compromised endocardial PI3K/AKT pathway activation, (3) an increased number of myofibroblasts and (4) increased fibrin levels, as well as bigger scars.Our data further revealed that lack of MyD88 signaling impairs CM behavior in the injured area.By endothelial cell-specific overexpression of myd88, we showed that it has an essential role in the endothelial response to injury by regulating neutrophil recruitment to the injury site as well as fibrosis.Mechanistically, we identified the chemokine gene cxcl18b as a target of the MyD88 signaling pathway that controls neutrophil recruitment (Fig. 7).Overall, these data highlight the beneficial role of MyD88 activation in the regenerative response to cardiac injury in zebrafish  and provide insights on how pathways activated very quickly after injury can shape the regenerative outcome.
While some studies suggested that MyD88 signaling promotes the regenerative process, others reported improved outcomes when MyD88 signaling was blocked 14,20,21 .Our data clearly show that MyD88 is a beneficial molecule for the regenerative process in zebrafish.The discrepancy with studies reporting detrimental effects of MyD88 signaling on regeneration mostly lies in the model used.Specifically, while inflammation is essential in the early stages of regeneration [9][10][11] , prolonged and excessive inflammation can lead to adverse outcomes 14,20 .In non-regenerative mouse models, inflammation tends to be uncontained; thus, limiting inflammation by blocking MyD88 signaling can enhance the regeneration potential [22][23][24][89][90][91] . Accordngly, blocking MyD88 function has been linked with protective effects in experimental settings (for example, endotoxin shock) after lipopolysaccharide administration 23,92,93 .Once again, these data reflect special cases where inflammation is uncontrolled and excessive.In addition, following myd88 overexpression in endothelial cells, we successfully reversed the reduced neutrophil number and the enhanced fibrosis phenotypes observed in cryoinjured myd88 −/− ventricles, indicating that MyD88 is required in endothelial cells for the regenerative response.However, myd88 overexpression in endothelial cells in cryoinjured myd88 +/+ ventricles did not lead to an increase in neutrophil numbers (Figs.1g and 5h) or an improvement in the fibrotic response (Figs.2c-g and 5a-f), indicating that MyD88 signaling is optimally tuned in WT conditions.Studies reported that endothelial-specific MyD88-deficient mice exhibit features of decreased inflammation 94 , while stimulation of the MyD88 pathway in aortic endothelial cells in rabbits promoted inflammation 95 . In lightof our data, it will be interesting to investigate in injured mammalian hearts which cells activate MyD88 signaling and the function of the MyD88 pathway in endocardial cells.
Myeloid cells serve as key activators of the MyD88 signaling pathway in response to injury or infection 22,[37][38][39][40] .The myd88 LOF mutant used in this study has also been used in the context of larval zebrafish tail fin regeneration, where it was reported that MyD88 controls neutrophil and macrophage recruitment to the wound 37 .In line with these findings, we observed reduced neutrophil and macrophage numbers in cryoinjured myd88 −/− ventricles.However, we do not anticipate major differences between myd88 −/− and WT zebrafish in the absence of an insult (injury or infection) that could trigger MyD88 pathway activation.As shown previously, early leukocyte hematopoiesis, migration, basal motility and phagocytosis are not affected by MyD88 deficiency 32,37 .In addition, transcriptomic analysis from uninfected myd88 −/− and WT embryos did not reveal any differences in immune-related genes other than myd88, which was explained by the lower stability of the mutant transcript 32 .In our study, we also did not observe any significant differences in neutrophil count between sham-injured myd88 −/− and myd88 +/+ ventricles (Extended Data Fig. 1f,g).Altogether, these data indicate that the observed phenotypes in cryoinjured myd88 −/− ventricles are due to the mutant's inability to activate the pathway in response to injury rather than to preexisting developmental defects.
We expanded our investigation beyond the conventional activators of the MyD88 signaling pathway, myeloid cells, to explore the impact of the loss of MyD88 function on other cell types.Our transcriptomic analysis pointed to endocardial cells, which in myd88 −/− ventricles display a fibrotic phenotype.Bulk RNA-seq analysis of endocardial cells revealed impairment in the activation of the MAPK and PI3K/AKT pathways in the myd88 −/− endocardium.We elected to focus on MyD88 function in the PI3K/AKT pathway because it was more strongly affected and has not been explored as extensively as the MAPK pathway in the endocardial response during cardiac regeneration in zebrafish 51,63,64 .For these and other studies, we then overexpressed myd88 in endothelial cells in cryoinjured myd88 −/− ventricles, which led to the reversal of the reduced neutrophil count and the enhanced fibrosis.Taken together, these findings underscore the importance of the MyD88 signaling pathway in endocardial cells and thus support the hypothesis that at least some of the myd88 −/− phenotypes after cardiac injury could originate mostly in the endocardium.Unlike most myeloid cells, which need to be recruited to the injured tissue, endocardial cells are inherently present and abundant in the heart 36,96 .Additionally, a previous study showed that MyD88 signaling in hematopoietic cells alone was not sufficient to trigger the inflammatory response in the injured mouse heart 97 .Specifically, myocardial infarction in Myd88 −/− and WT mice reconstituted with WT bone marrow cells led to no differences in cytokine or chemokine levels 97 .Furthermore, endothelial cells activate immune-regulatory programs, secrete cytokines and chemokines, and initiate immune cell recruitment 52,55,80 .They express the tlr and myd88 genes 52,53 and at least a subgroup of endothelial cells, vascular endothelial cells, rely exclusively on MyD88 for TLR activation 98 .Additionally, signaling cascades commonly activated by MyD88, such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and PI3K/ AKT, get activated in endothelial cells 69,99 .These studies, together with our data, emphasize the pivotal role of early MyD88 signaling activation in endocardial cells and support the hypothesis that the fibrotic phenotype in cryoinjured myd88 −/− ventricles initiates in the endocardium.
During cardiac development and regeneration, the endocardium has a crucial role in signaling to CMs 55,100 , while the ECM supports CM behavior [101][102][103] .Given the altered inflammatory and fibrotic environment observed in cryoinjured myd88 −/− ventricles, along with the impaired endocardial response preceding CM appearance in the injured area, we investigated CM behavior.Our findings revealed decreased CM proliferation and reduced CM protrusion length in cryoinjured myd88 −/− ventricles.We hypothesize that the myd88 −/− CM environment is not sufficiently permissive for CM protrusions to extend as they do in myd88 +/+ siblings.However, we cannot exclude the possibility that CMs also activate MyD88 signaling, as activity of NF-κB, a MyD88 target, has also been reported in CMs 104 .Unfortunately, our scRNA-seq analysis did not manage to capture a CM population; similar challenges with CM populations have been reported in other transcriptomic studies 36,105 .In addition, it is probable that epicardial cells also activate MyD88 signaling during cardiac regeneration in zebrafish.The number of superficially localized αSMA + cells, presumably of epicardial, fibroblast and perivascular origin, was also elevated in cryoinjured myd88 −/− ventricles (Extended Data Fig. 2e).To draw more definitive conclusions about the role of MyD88 in CMs, epicardial cells and other cardiac cell populations, additional cell type-specific studies will need to be performed.
Our data also reveal that the expression of Cxcl18b, a neutrophil chemoattractant produced in the endocardium, is induced after  MyD88 activation.Notably, we observed that cxcl18b overexpression partially rescued the reduced neutrophil count in cryoinjured myd88 −/− ventricles (Fig. 6e,f).Neutrophil recruitment via Cxcl18b was previously reported not to be dependent on the dosage of Cxcl18b because receptor saturation might occur or neutrophils might not be able to get stimulated further 83 .Our inability to fully rescue neutrophil numbers with cxcl18b overexpression might, at least in part, be due to these issues or the involvement of other chemoattractants.We also generated a cxcl18b full locus deletion allele and showed that homozygous mutants displayed significantly reduced neutrophil numbers at 96 hpci.As there was no clear difference in neutrophil count at 24 hpci between cryoinjured cxcl18b −/− and cxcl18b +/+ ventricles, we again speculate that Cxcl18b is one of several chemokines secreted after injury and required for neutrophil recruitment 83 , and that its absence alone is not sufficient to trigger a difference at early time points.
In summary, our findings highlight the crucial role of MyD88 signaling in cardiac regeneration.We propose that MyD88 regulates both the inflammatory and fibrotic responses and that its early activation in the endocardium is vital for successful regeneration.Additionally, we identified the chemokine Cxcl18b as a target of MyD88 signaling that contributes to neutrophil recruitment.Gaining a deeper understanding of the early innate immune mechanisms activated in the injured heart is of vital importance because they can influence subsequent processes and thereby impact the regenerative outcome.In addition, a comprehensive understanding of the MyD88 pathway's contribution to the regenerative response will determine whether targeting a molecule of the innate immune system could be considered as a treatment target to limit pathogenesis in cardiovascular diseases.

Zebrafish husbandry and handling
Zebrafish larvae were raised under standard conditions.Adult fish were maintained in 3.5-l tanks at a stock density of ten fish per liter with the following parameters: water temperature, 27-27.5 °C; light-dark cycle, 14:10; pH, 7.0-7.5;conductivity, 750-800 μS cm −2 .Zebrafish were fed 3-5 times a day, depending on age, with granular and live food (Artemia salina).Health monitoring was performed twice a year.All procedures performed on animals conformed to the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes and were approved by the Animal Protection Committee (Tierschutzkommission) of the Regierungspräsidium Darmstadt (reference nos.B2/1218 and B2/1229).

Generation of zebrafish mutant and transgenic lines
Sequence analysis for the generation of new lines was performed using the ApE software (v.2.0.61).To generate the cxcl18b bns683 full locus deletion allele, the CRISPR-Cas9 technology was used, as described previously [110][111][112] .The single-guide RNAs (sgRNAs) GGAAAGTTACAAGGAAATGC and GGAAGTTTGGATGATTCTAA were designed with a CRISPR design tool (https://www.crisprscan.org/)targeting sequences upstream and downstream of the 5′ and 3′ untranslated regions, respectively.sgRNAs were transcribed using a MEGAshortscript T7 Kit (Thermo Fisher Scientific) and later purified with the RNA Clean & Concentrator Kit (Zymo Research).Then, 50 pg of each sgRNA were coinjected with 100 pg of cas9 mRNA into zebrafish one-cell-stage WT embryos.Injected embryos were raised to adulthood and later screened for founder In endocardial cells, PI3K/AKT pathway activation and fibrosis are controlled by the MyD88 signaling pathway.Neutrophil count is also partially affected by the levels of the endocardial chemokine Cxcl18b.Immune cells also activate a MyD88-mediated response leading to several processes, including the recruitment of more immune cells and the manifestation of an inflammatory response.b, In myd88 −/− injured tissues, the MyD88 signaling pathway is not activated and thus, endocardial cells exhibit decreased activation of the PI3K/AKT pathway.myd88 −/− injured tissues also exhibit decreased levels of the endocardial chemokine gene cxcl18b and an increase in features related to fibrosis.In addition, the neutrophil count appears significantly reduced.As the fibrotic and the immune responses are affected, other processes essential for successful regeneration are also impaired, including revascularization and CM proliferation.
To generate the Tg( fli1a:myd88,EGFP) bns703 line, in which a bidirectional fli1a promoter drives the expression of both myd88 and EGFP, we modified the fli1a:mCherry,EGFP construct provided by C. Helker.The myd88 coding sequence was cloned and subsequently used to replace the mCherry coding sequence.Cloning was performed using the In-Fusion HD Cloning Kit (Takara Bio).Then, 10 pg of the final construct were coinjected with 25 pg of Tol2 mRNA into zebrafish one-cell-stage WT embryos.Injected embryos positive for EGFP were raised to adulthood and later screened for founder identification.
To generate the Tg(hsp70l:loxP-TagBFP-loxP-cxcl18b-t2a-mCherry) bns660 line, the hsp70l:loxP-TagBFP-loxP-il11ra-t2a-mCherry construct was modified 59 .The cxcl18b coding sequence was cloned and subsequently used to replace the il11ra coding sequence.Cloning was performed using the In-Fusion HD Cloning Kit.Then, 10 pg of the final construct were coinjected with 25 pg of Tol2 mRNA into zebrafish one-cell-stage WT embryos.Injected embryos were heat-shocked and embryos positive for TagBFP were raised to adulthood and later screened for founder identification.To induce recombination, 12.5 pg of Cre mRNA were injected into Tg(hsp70l :loxP-TagBFP-loxP-cxcl18b-t2a-mCherry) one-cell-stage embryos.Cre mRNA-injected embryos were heat-shocked and embryos positive for mCherry were raised to adulthood.

Cardiac cryoinjury and heat shock treatments
Cardiac cryoinjury was performed in adult zebrafish hearts as described previously [3][4][5] .Zebrafish were briefly anesthetized with tricaine and transferred on a wet sponge with their ventral side up.A small incision was made on the chest area exposing the heart.A cryoprobe precooled in liquid nitrogen was applied to the ventricular apex until thawing.Cryoinjured fish were transferred into fresh system water and left to recover.Heat shock treatments to induce the expression of the hsp70l-driven transgene Tg(hsp70l:loxP-TagB FP-loxP-cxcl18b-t2a-mCherry) were performed by incubating the adult fish or the embryos in preheated system or egg water (39 °C) for 1 h.Several heat shocks were performed as described in the corresponding experimental plans.It is important to note that heat shock alone, as a stress response, can promote an immune response, including the influx of inflammatory cells 113,114 , potentially explaining the discrepancy in neutrophil count in cryoinjured ventricles observed with (Fig. 6f) and without (Fig. 1g) heat shock treatments.

Histological analysis and imaging
For the histological analysis, zebrafish hearts were fixed in 4% paraformaldehyde for 1 h at room temperature and then preserved overnight in 30% (w/v) sucrose solution prepared in 1× PBS at 4 °C.Hearts samples were embedded in O.C.T. (Tissue-Tek) and stored at −80 °C until further use.Eight and 50-μm-thick cryosections were collected on SuperFrost Plus slides (Thermo Fisher Scientific) using the Leica CM1950 cryostat and stored at −20 °C.
For AFOG staining, slides were thawed for 15 min at room temperature, rinsed twice with 0.1% Triton X-100 in 1× PBS to remove O.C.T., incubated in Bouin's solution for 2 h at 60 °C and then stained according to the manufacturer's instructions (AFOG staining kit, BioGnost) and without hematoxylin solution 59 .Stained slides were imaged using a Nikon SMZ25 stereo microscope coupled with a Nikon Digital Sight DS-Ri1 camera.
For immunofluorescence staining, slides were thawed for 15 min at room temperature, rinsed twice with 0.1% Triton X-100 in 1× PBS to remove O.C.T. and permeabilized with 0.5% Triton X-100 in 1× PBS for 20 min (2 h for 50-μm-thick cryosections) at room temperature.Cryosections were then incubated in blocking buffer solution (1× PBS, 2% (v/v) goat serum, 0.2% Triton X-100 and 1% dimethyl sulfoxide) for 1 h at room temperature.Then, cryosections were incubated with primary antibodies in blocking buffer solution overnight at 4 °C, rinsed three times every 10 min with 0.1% Triton X-100 in 1× PBS and incubated with secondary antibodies in blocking buffer solution for 3 h at room temperature.Lastly, immunostained cryosections were rinsed three times every 10 min with 0.1% Triton X-100 in 1× PBS, incubated in DAPI (1:10,000 dilution, Sigma-Aldrich) for 5 min at room temperature and mounted with fluorescence mounting medium (cat.no.S3023, Agilent Dako) for imaging.MEF2, PCNA, pAkt and pERK immunostaining was performed as described previously 73 .MEF2 and pERK immunostaining was supplemented with an additional step after O.C.T. removal, which consisted of antigen retrieval in 10 mM sodium citrate buffer with 0.05% (v/v) Tween-20 (all from Sigma-Aldrich), pH 6.0, for 7 min at 95 °C.

Quantification and statistical analysis
Quantification was done in two or three non-consecutive sections per ventricle and with the ZEN Blue Edition software.Quantification of the mpx:GFP + , mpeg1:EGFP + and Mpx + cell numbers was performed in the injured tissue and in peripheral border zone areas (100 μm).The mpx:GFP + cell number analysis for the myd88 +/− control condition (Fig. 6e,f) was performed separately.Quantification of the total αSMA + cell number was performed in the injured tissue, and of the intraventricular αSMA + cell number was performed in the injured tissue excluding the superficial/peripheral αSMA + cells (that is, quantification was based on cell localization and not on the presence of an additional cell marker).Quantification of intraventricularly localized αSMA + cells did not include double-positive αSMA + krt4:EGFP + cells Article https://doi.org/10.1038/s44161-024-00538-5 for the following reasons: (1) the endocardium has not fully extended into the injured tissue at 96 hpci (the time point of our analysis), as indicated by krt4:EGFP + expression, and (2) even though αSMA + cells can derive from the endocardium, it is not known how long they retain the expression of endocardial markers.The αSMA + cell number analysis displayed in Fig. 2d is the result of two independent experiments, each of which contained samples of both genotypes from the same batch.For the endocardial pAkt activation and proliferation analysis, the percentage of pAkt + and PCNA + endocardial cells was calculated as a ratio of the total number of endocardial cells (krt4:EGFP + Fli1 + ) in a 50-μm wide area on the basal-most side of the injured tissue, as described previously 55 .For the krt4:EGFP + , Cdh5 + and pERK + cell area analysis, the fluorescent area within the injured tissue was measured and then divided by the total injured tissue area.For the CM dedifferentiation and proliferation analysis, the percentage of N2.261 + and PCNA + CMs was calculated as a ratio of the total number of CMs (MEF2 + ) in peripheral border zone areas (100 μm).CM protrusions and protrusion lengths were measured in two non-consecutive 50-μm-thick cryosections with the largest injured area from each heart.The start of the protrusions was defined as the injury border and the end of the protrusions as the point where the F-actin signal disappeared in the injured tissue, as described previously 77 .All protrusions measured were extending toward the injured tissue.For the cEC proliferation analysis, the percentage of PCNA + cECs was calculated as a ratio of the total number of cECs (-0.8flt1:RFP + ) in the injured tissue and in peripheral border zone areas (200 μm).For the coronary vessel coverage analysis, the percentage of the fluorescence intensity was calculated as a ratio of the background fluorescence in the injured tissue using ImageJ (v.1.53c)in wholemount images.For the scar area analyses, scar areas were selected based on the combined occurrence of collagen and fibrin within the ventricle.The average of the ratio of the 2-3 biggest scar areas to the total ventricular areas were calculated using ImageJ (v.1.53c).Percentages of scar areas (relative to ventricular areas) were then grouped based on their size.Fibrin and collagen measurements were performed as described previously 62 .
All statistical analyses were performed in GraphPad Prism (v.9).Distribution of data in each group was assessed using the Shapiro-Wilk normality test.Data that were normally distributed were further analyzed with a two-tailed Student's t-test.Data that were not normally distributed were further analyzed with a two-tailed Mann-Whitney U-test.The significance level was set to 0.05 for all tests.The exact P values are indicated in the figures.The error bars in the figures represent the mean ± s.d.

Tissue dissociation and cell sorting
For the scRNA-seq experiment, cardiac cells were isolated from a pool of four myd88 +/+ and four myd88 −/− ventricles, and for the endocardial bulk RNA-seq experiment from a pool of four ET(krt4:EGFP); myd88 +/+ and four ET(krt4:EGFP); myd88 −/− ventricles for each sample.Cell isolation was performed according to the manufacturer's instructions (Pierce Primary Cardiomyocyte Isolation Kit, cat.no.88281, Thermo Fisher Scientific) and with the following modifications: incubation was performed at 30 °C for 20 min, followed by resuspension in 1× Hanks' Balanced Salt Solution (cat.no.14175053, Gibco) with 0.25% BSA.The cell suspension was passed through a round bottom polystyrene test tube fitted with a 35-μm nylon mesh filter cap (Falcon, cat.no.352235, Corning).DAPI (cat.no.D954, Sigma-Aldrich) was added before sorting.For the scRNA-seq experiment, resuspended cells were sorted using a BD FACSAria III Cell Sorter (BD Biosciences) equipped with a 100-μm nozzle and at an instrument pressure setting of 20 psi.Dead cells were excluded using DAPI excited by a 30-mW 405-nm laser paired with a 450/50-nm band-pass filter.For the endocardial bulk RNA-seq experiment, resuspended cells were sorted using an Invitrogen Bigfoot Spectral Cell Sorter (Thermo Fisher Scientific) equipped with a 100-μm nozzle tip and at an instrument pressure setting of 20 psi.Dead cells were excluded using DAPI excited by a 100-mW 355-nm laser paired with 455/14-nm band-pass filter.EGFP fluorescence was measured with a 100-mW 488-nm excitation paired with a 530/30-nm band-pass filter.Sorted EGFP + cells were resuspended in ice-cold QIAzol Lysis Reagent (QIAGEN), flash-frozen in liquid nitrogen and kept at −80 °C until RNA extraction.Cytometric data were recorded using the FACSDiva software v.8.0.1 (BD Biosciences) and the Sasquatch software v.1.19.2 (Thermo Fisher Scientific).The .fcs files were analyzed using FlowJo v.10.8.1 (BD Life Sciences).

scRNA-seq analysis
For the scRNA-seq experiment, cells were counted with a Moxi cell counter and diluted according to the manufacturer's protocol to obtain 10,000 single-cell data points per sample.Each sample was run separately on a lane in a Chromium controller with the Chromium Next GEM Single Cell 3′ Reagent Kits v.3.1 (10x Genomics).scRNA-seq library preparation was done using a standard protocol.Sequencing was done on NextSeq 2000 system and raw reads were aligned against the zebrafish genome (DanRer11) and counted using STARsolo 115 followed by secondary analysis in annotated data format.Preprocessed counts were further analyzed using Scanpy 116 .Basic cell quality control was conducted by taking the number of detected genes and mitochondrial content into consideration.We removed 32 cells that did not express more than 300 genes or had a mitochondrial content greater than 10%.Furthermore, we filtered 7,823 genes if they were detected in fewer than 30 cells (<0.01%).The raw counts per cell were normalized to the median count over all cells and transformed into log space to stabilize variance.We initially reduced the dimensionality of the dataset using principal component analysis, retaining 50 principal components.Final data visualization was done using the scVelo 117,118 and CELLxGENE packages.

Bulk RNA-seq analysis
For bulk RNA-seq of endocardial cells (Fig. 3a), RNA was isolated from the fluorescence-activated cell-sorted endocardial cells using the miRNeasy Micro Kit (QIAGEN) combined with on-column DNase digestion (RNase-free DNase Set, QIAGEN) to avoid contamination by genomic DNA (gDNA).Subsequent RNA quality control analysis, complementary DNA (cDNA) preparation and sequencing were performed by Novogene.mRNA was purified from total RNA with poly-T oligo-attached magnetic beads, went through fragmentation and cDNA was synthesized.Trimmomatic 119 was used to trim the reads.Reads longer than 15 nucleotides after trimming were kept and aligned to the Ensembl zebrafish genome v.danRer11 (Ensembl release 109) with STAR aligner (v.2.7.10a) 115 .Alignments were filtered with the Picard tool (v.3.0.0) to remove duplicates, multimapping, ribosomal or mitochondrial reads.Gene counts were generated using the featureCounts tool (v.2.0.4), taking all reads overlapping annotated exons into account and excluding those overlapping multiple genes 120 .Finally, the raw count matrix was normalized and contrasts were created and analyzed using DESeq2 (v.1.36.0) 121 .Genes were classified as significantly differentially expressed at an average count greater than five, multiple testing P adj < 0.05 and −0.585 < log 2 fold change > 0.585.Heatmaps with DEGs were obtained using the WIlsON 122 .To perform GSEA, we used the Python package gseapy 123 .As gene sets, we used 162 custom Danio rerio mapped gene sets from reactome and 3,023 gene sets from Gene Ontology.Gene ranking was performed according to the DESeq-derived P adj multiplied by the direction (+ or −) of the log 2 fold change.The GSEA analysis was run for gene sets with a min_size = 5, max_size = 1,000 and permutation_num = 1,000.From the resulting lists, representative sets were selected.
For bulk RNA-seq of untouched ventricles and injured tissues (Fig. 6a), RNA was isolated from three pooled ventricles or three pooled injured tissues per sample using the miRNeasy Micro Kit (QIAGEN) combined with on-column DNase digestion (RNase-free Article https://doi.org/10.1038/s44161-024-00538-5 DNase Set, QIAGEN) to avoid contamination by gDNA.Two biological replicates were prepared for each condition.RNA and library preparation integrity were verified with LabChip Gx Touch 24 Analyzer (PerkinElmer).400 ng of total RNA was used as input for the VAHTS Stranded mRNA-seq Library Preparation V6 according to the manufacturer's protocol (Vazyme).Sequencing was performed on an NextSeq 500 instrument (Illumina) using v2 chemistry with 1 × 75-bp single end setup.Trimmomatic v.0.39 was used to trim reads after a quality drop below a mean of Q15 in a window of five nucleotides, keeping only filtered reads longer than 15 nucleotides 119 .Reads were aligned against the Ensembl zebrafish genome v.danRer11 (Ensembl release 99) with STAR v.2.7.3a 115 .Alignments were filtered with Picard v.2.21.7 to remove duplicates, multimapping, ribosomal or mitochondrial reads.Gene counts were established with featureCounts v.1.6.5 aggregating reads overlapping exons, excluding those overlapping multiple genes 120 .The raw count matrix was normalized with DESeq2 v. 1.26.0  (ref.121).Contrasts were created with DESeq2 based on the raw count matrix.Genes were classified as significantly differentially expressed at average count greater than five, multiple testing P adj < 0.05 and −0.585 < log 2 fold change > 0.585.Heatmaps with DEGs were obtained using WIlsON 122 .

RT-qPCR
For RT-qPCR, RNA was extracted from three pooled cryoinjured tissues (1 hpci) and ventricles (96 hpci) (Fig. 6c) or from single larvae (Extended Data Fig. 9a and Extended Data Fig. 10c,f) or from single cryoinjured ventricles (96 hpci) (Extended Data Fig. 9a) per biological replicate using the TRIzol Reagent (Thermo Fisher Scientific) using the phenol-chloroform extraction protocol.Total RNA was purified with the RNA Clean & Concentrator Extraction Kit (Zymo Research) according to the manufacturer's instructions.At least 250 ng of total RNA per sample was reverse-transcribed with the Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) according to the manufacturer's instructions.All reactions were performed with three technical replicates using the SYBR Green PCR Master Mix (Thermo Fisher Scientific) on the CFX Connect Real-Time System (CFX Manager 3.1, Bio-Rad Laboratories) and with the following program: preamplification at 95 °C for 7 min followed by 39 cycles of amplification at 95 °C for 5 s and 60 °C for 20 s, using a melting curve from 60 to 92 °C with an increment of 1.0 °C every 5 s.Gene mRNA levels were normalized against the rpl13a mRNA levels and fold changes were calculated using the 2− ΔΔ Ct method.The RT-qPCR primer sequences are listed in Supplementary Table 2.The average Ct values of the RT-qPCR are listed in Supplementary Table 3.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Fig. 7 |
Fig. 7 | Proposed model for the role of MyD88 in fibrosis and neutrophil recruitment following cardiac cryoinjury in zebrafish.a, TLRs (and IL-1Rs) recruit the adaptor molecule MyD88 upon ligand interaction.MyD88 in turn initiates signal transduction, which leads to the activation of several processes.In endocardial cells, PI3K/AKT pathway activation and fibrosis are controlled by the MyD88 signaling pathway.Neutrophil count is also partially affected by the levels of the endocardial chemokine Cxcl18b.Immune cells also activate a MyD88-mediated response leading to several processes, including the recruitment of more immune cells and the manifestation of an inflammatory